Process for oxidising aromatic compounds

ABSTRACT

Process of oxidising a substrate which is a halo aromatic compound, which process comprises oxidizing said substrate with a monooxygenase enzyme. The enzyme may be P450cam. The process may be carried out in cells, animals or plants.

The invention relates to a process for enzymatically oxidizinghalogenated aromatic compounds.

Chlorinated aromatic compounds such as the chlorobenzene andpolychlorinated biphenyls (PCBs) are among the most wide-spread organiccontaminants in the environment due to their common application assolvents, biocides, and in the heavy electrical industry. They are alsosome of the most problematic environmental pollutant, not only becauseof the health hazards (lipid solubility and hence accumulation in fattytissues, toxicity and carcinogenicity) but also because of their slowdegradation in the environment.

Whilst microorganisms have shown extraordinary abilities to adapt andevolve to degrade most of the organic chemicals released into theenvironment, the most chemically inert compounds such as PCBs do persistfor two main reasons. First, these compounds have very low solubility inwater and therefore their bioavailability is low. Research into thisproblem has focussed on the use of detergents and other surfactants toenhance their solubility and bioavailability. Second, these compoundsrequire activation by enzymatic oxidation or reduction, and it can takea long time for the necessary genetic adaptations by microorganisms tooccur, and even then the organisms may not be stable and viable.

We have now found, according to the present invention, that amonoxygenase, in particular P450_(cam) and its physiological electrontransfer partners putidaretoxin and putidaretoxin reductase, can be usedto oxidise halogenated aromatic compounds. Also mutants of themonoxygenase with substitutions in the active site have enhancedoxidation activity. Thus suitable monoxygenases can be expressed inmicroorganisms, animals and plants which are going to be used to oxidisethe halogenated aromatic compounds.

Accordingly the present invention provides a process for oxidizing asubstrate which is a halo aromatic compound, which process comprisesoxidizing said substrate with a monooxygenase enzyme.

The process may be carried out in a cell that expresses:

(a) the enzyme

(b) an electron transfer reductase; and

(c) an electron transfer redoxin

The halo aromatic compound is typically a benzene or biphenyl compound.The benzene ring is optionally fused and can be substituted. The halogenis typically chlorine. In many cases there is more than one halogen atomin the molecule, typically 2 to 5 or 6, for example 3. Generally 2 ofthe halogen atoms will be ortho or para to one another. The compound mayor may not contain an oxygen atom such as a hydroxy group, an aryloxygroup or a carboxy group. The compound may or may not be chlorophenol ora chlorophenoxyacetic compound.

Specific compounds which can be oxidised by the process of the presentinvention include 1,2; 1,3- and 1,4-dichlorobenzene, 1,2,4; 1,2,3- and1,3,5-trichlorobenzene, 1,2,4,5- and 1,2,3,5-tetrachlorobenzene,pentachlorobenzene, hexachlorobenzene, 3,3′-dichlorobiphenyl and2,3,4,5,6- and 2,2′,4,5,5′-pentachlorobiphenyl.

Other compounds which can be oxidised by the process includerecalcitrant halo aromatic compounds, especially dioxins and halogenateddibenzofurans, and the corresponding compounds where one or both oxygenatoms is/are replaced by sulphur, in particular compounds of theformula:

which possess at least one halo substituent, such as dioxin itself,2,3,7,8-tetrachlorodibenzioxin.

The oxidation typically gives rise to 1,2 or more oxidation products.These oxidation products will generally comprise 1 or more hydroxylgroups. Generally, therefore, the oxidation products are phenols whichcan readily be degraded. It is particularly noteworthy thatpentachlorobenzene and hexachlorobenzene can be oxidised in this waysince they are very difficult to degrade. In contrast the correspondingphenols can be readily degraded by a variety of Pseudomonas and otherbacteria. The atom which is oxidized is generally a ring carbon.

The enzyme is typically a natural monooxygenase or a mutant thereof. Thenatural monooxygenase is generally a prokaryotic or eukaryotic enzyme.Typically it is a haem-containing enzyme and/or a P450 enzyme. Themonooxygenase may or may not be a TfdA (2,4-dichlorophenoxy)acetate/α-KG dioxygenase. The monooxygenase is generally ofmicroorganism (e.g. bacterial), fungal, yeast, plant or animal origin,typically of a bacterium of the genus Pseudomonas. These organisms aretypically soil, fresh water or salt water dwelling. In the case of amutant monooxygenase the non-mutant form may or may not be able tooxidize the substrate.

The monooxygenase typically has a coupling efficiency of at least 1%,such as at least 2%, 4%, 6% or more. The monooxygenase typically has aproduct formation rate of at least 5 min⁻¹, such as at least 8, 10, 15,20, 25, 50, 100, 150 min⁻¹ or more. The coupling efficiency or productformation rate is typically measured using any of the substrates orconditions mentioned herein. Thus they are typically measured in the invitro conditions described in Example 2, in which case the relevantmonooxygenase, reductase and redoxin would be present instead of, but atthe same concentration as, P450_(cam), putidaretoxin reductase andputidaretoxin.

The mutant typically has at least one mutation in the active site. Apreferred mutant comprises a substitution of an amino acid in the activesite by an amino acid with a less polar side chain. Thus the amino acidis typically substituted with an amino acid which is above it in Table1.

TABLE 1 HYDROPATHY SCALE FOR AMINO ACID SIDE CHAINS Side ChainHydropathy Ile 4.5 Val 4.2 Leu 3.8 Phe 2.8 Cys 2.5 Met 1.9 Ala 1.8 Gly−0.4 Thr −0.7 Ser −0.8 Trp −0.9 Tyr −1.3 Pro −1.6 His −3.2 Glu −3.5 Gln−3.5 Asp −3.5 Asn −3.5 Lys −3.9 Arg −4.5

An amino acid ‘in the active site’ is one which lines or defines thesite in which the substrate is bound during catalysis or one which linesor defines a site through which the substrate must pass before reachingthe catalytic site. Therefore such an amino acid typically inateractswith the substrate during entry to the catalytic site or duringcatalysis. Such an interaction typically occurs through an electrostaticinteraction (between charged or polar groups), hydrophobic interaction,hydrogen bonding or van der Waals forces.

The amino acids in the active site can be identified by routine methodsto those skilled in the art. These methods include labelling studies inwhich the enzyme is allowed to bind a substrate which modifies(‘labels’) amino acids which contact the substrate. Alternatively thecrystal structure of the enzyme with bound substrate can be obtained inorder to deduce the amino acids in the active site.

The monooxygenase typically has 1, 2, 3, 4 or more other mutations, suchas substitutions, insertions or deletions. The other mutations may be inthe active site or outside the active site. Typically the mutations arein the ‘second sphere’ residues which affect or contact the position ororientation of one or more of the amino acids in the active site. Theinsertion is typically at the N and/or C terminal and thus the enzymemay be part of a fusion protein. The deletion typically comprises thedeletion of amino acids which are not involved in catalysis, such asthose outside the active site. The monooxygenase may thus comprise onlythose amino acids which are required for oxidation activity.

The other mutations in the active site typically alter the positionand/or conformation of the substrate when it is bound in the activesite. The mutation may make the site on the substrate which is to beoxidized more accessible to the haem group. Thus the mutation may be asubstitution to an amino acid which has a smaller or larger, or more orless polar, side chain.

The other mutations typically increase the stability of the protein, ormake it easier to purify the protein. They typically prevent thedimerisation of the protein, typically by removing cysteine residuesfrom the protein (e.g. by substitution of cysteine at position 334 ofP450_(cam), or at an equivalent position in a homologue, preferably toalanine). They typically allow the protein to be prepared in solubleform, for example by the introduction of deletions or a poly-histidinetag, or by mutation of the N-terminal membrane anchoring sequence. Themutations typically inhibit protein oligomerisation, such asoligomerisation arising from contacts between hydrophobic patches onprotein surfaces.

Typically the mutant monoxygenase is at least 70% homologous to anatural monooxygenase on the basis of amino acid identity.

Any of the homologous proteins mentioned herein are typically at least70% homologous to a protein or at least 80 or 90% and more preferably atleast 95%, 97% or 99% homologous thereto over at least 20, preferably atleast 30, for instance at least 40, 60 or 100 or more contiguous aminoacids. The contiguous amino acids may include the active site. Thishomology may alternatively be measured not over contiguous amino acidsor nucleotides but over only the amino acids in the active site.

The monoxygenase is preferably:

(i) P450_(cam),

(ii) a naturally occurring homologue of (i),

(iii) a mutant of (i) or (ii).

Typically (i) is any allelic variant of P450_(cam) of Pseudomonas putida(e.g. of the polypeptide sequence shown in SEQ ID No. 2). Typically (ii)is a species homologue of (i) which has sequence homology with (i), andis typically P450_(BM-3) of Bacillus megaterium (e.g. the polypeptidesequence shown in SEQ ID No. 4 and the nucelotide sequence is shown inSEQ ID NO: 3), P450_(terp) of Pseudomonas sp, P450_(eryF) ofSaccharopollyspora erythraea, or P450 105 D1 (CYP105) of Streptomycesgriseus strains.

The active site of (ii) or (iii) may be substantially the same as theactive site of (i) or any of the mutants of (i) mentioned herein. Thusthe site may comprise the same amino acids in substantially the samepositions.

Typically in (iii) amino acid 96 of P450_(cam), or the equivalent aminoacid in a homologue, has been changed to an amino acid with a less polarside chain.

The ‘equivalent’ side chain in the homologue is one at the homologousposition. This can be deduced by lining up the P450_(cam), sequence andthe sequence of the homologue based on the homology between the twosequences. The PILEUP, BLAST and BESTFIT algorithms can be used to lineup the sequences (for example as described in Altschul S. F. (1993) JMol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10and (Devereux et al (1984) Nucleic Acids Research 12, p387-395)). Thesealgorithms can also be used to calculate the levels of homologydiscussed herein (for example on their default settings). The equivalentamino acid will generally be in a similar place in the active site ofthe homologue as amino acid 96 in P450_(cam).

The discussion below provides examples of the positions at whichsubstitutions may be made in P450_(cam). The same substitutions may bemade at equivalent positions in the homologues. Standard nomenclature isused to denote the mutations. The letter of the amino acid present inthe natural form is followed by the position, followed by the amino acidin the mutant. To denote multiple mutations in the same protein eachmutation is listed separated by hyphens. The mutations discussed belowusing this nomenclature specify the natural amino acid in P450_(cam),but it is to be understood that the mutation could be made to ahomologue which has a different amino acid at the equivalent position.

An additional mutation is typically an amino acid substitution at aminoacid 87, 98, 101, 185, 244, 247, 248, 296, 395, 396 or a combination ofthese, for example as shown in table 2.

The following combinations of substitutions are preferred:

(i) Substitution at position 87 to amino acids of different side-chainvolume, such as substitutions (typically of F) to A, L, I and W,combined with substitutions at position 96 to amino acids of differentside-chain volume such as (typically Y to) A, L, F, and W. Thesecombinations alter the space available in the upper part of thesubstrate pocket compared to the wild-type enzyme, for example, fromY96W-F87W (little space) to Y96A-F87A (more space), as well as thelocation of the space, for example from one side in Y96F-F87A to theother in Y96A-F87W.

(ii) Substitution at position 96 to F combined with substitutions atpositions 185 and 395. Both T185 and I395 are at the upper part of thesubstrate pocket, and substitution with A creates more space whilesubstitution with F will reduce the space available and push thesubstrate close to the haem.

(iii) Substitutions at position 96 to A, L, F, and W combined withsubstitutions at residues closer to the haem including at 101, 244, 247,295, 296 and 396 to A, L, F, or W. These combinations will create orreduce space in the region of the different side-chains to offerdifferent binding orientations to substrates of different sizes. Forexample, the combinations Y96W-L244A and Y96L-V247W will offer verydifferent pockets for the binding of the substrate.

(iv) Triple substitutions at combinations of positions 87, 96, 244, 247,295, 296, 395 and 396 with combinations of A, L, F, and W. The aim is tovary the size and shape of the hydrophobic substrate binding pocket. Forexample, the Y96A-F87A-L244A combination creates more space compared tothe Y96F-F87W-V396L combination, thus allowing larger substrates to bindto the former while restricting the available binding orientations ofsmaller substrates in the latter. The combinations Y96F-F87W-V247L andY96F-F87W-V295I have comparable substrate pocket volumes, but thelocations of the space available for substrate binding are verydifferent. The combination Y96F-F87L-V247A has a slightly largerside-chain volume at the 96 position than the combinationY96L-F87L-V247A, but the L side-chain at the 96 position is much moreflexible and the substrate binding orientations will be different forthe two triple mutants.

(v) The mutants with four or five substitutions were designed withsimilar principles of manipulating the substrate volume, the differentflexibility of various side-chains, and the location of the spaceavailable in the substrate pocket for substrate binding so as to effectchanges in selectivity of substrate oxidation.

Mutations are generally introduced into the enzyme by using methodsknown in the art, such as site directed mutagenesis of the enzyme, PCRand gene shuffling methods or by the use of multiple mutagenicoligonucleotides in cycles of site-directed mutagenesis. Thus themutations may be introduced in a directed or random manner. Typicallythe mutagenesis method produces one or more polynucleotides encoding oneor more different mutants. In one embodiment a library of mutantoligonucleotides is produced which can be used to produce a library ofmutant enzymes.

The process is typically carried out in the presence of the naturalcofactors of the monooxygenase. Thus typically in addition to the enzyme(a) and the substrate the process is carried out in the presence of anelectron transfer reductase (b), an electron transfer redoxin (c),cofactor for the enzyme and an oxygen donor. In this system the flow ofelectrons is generally: cofactor→(b)→(c)→(a).

(b) is generally an electron transfer reductase which is able to mediatethe transfer of electrons from the cofactor to (c), such as a naturallyoccurring reductase or a protein which has homology with a naturallyoccurring reductase, such as at least 70% homology, or a fragment of thereductase or homologue. (b) is typically a reductase of any of theorganisms mentioned herein, and is typically a flavin dependentreductase, such as putidaredoxin reductase.

(c) is generally an electron transfer redoxin which is able to mediatethe transfer of electrons from the cofactor to (a) via (b). (c) istypically a naturally occurring electron transfer redoxin or a proteinwhich has homology with a naturally occuring electron transfer redoxin,such as at least 70% homology; or a fragment of the redoxin orhomologue. (c) is typically a redoxin of any of the organisms mentionedherein. (c) is typically a two-iron/two sulphur redoxin, such asputidaredoxin.

The cofactor is any compound capable of donating an electron to (b),such as NADH. The oxygen donor is any compound capable of donatingoxygen to (a), such as dioxygen.

Typically (a), (b) and (c) are present as separate proteins; howeverthey may be present in the same fusion protein. Typically only two ofthem, preferably (b) and (c), are present in the fusion protein.Typically these components are contiguous in the fusion protein andthere is no linker peptide present.

Alternatively a linker may be present between the components. The linkergenerally comprises amino acids that do not have bulky side chains andtherefore do not obstruct the folding of the protein subunits.Preferably the amino acids in the linker are uncharged. Preferred aminoacids in the linker are glycine, serine, alanine or threonine. In oneembodiment the linker comprises the sequenceN-Thr-Asp-Gly-Gly-Ser-Ser-Ser-C (SEQ ID NO:6). The linker is typicallyfrom at least 5 amino acids long, such as at least 10, 30 or 50 or moreamino acids long.

In the process the concentration of (a), (b) or (c) is typically from10⁻⁸ to 10⁻²M, preferably from 10⁻⁶ to 10⁻⁴M. Typically the ratio ofconcentrations of (a):(b) and/or (a):(c) is from 0.1:01 to 1:10,preferably from 1:0.5 to 1:2, or from 1:0.8 to 1:1.2. Generally theprocess is carried out at a temperature and/or pH at which the enzyme isfunctional, such as when the enzyme has at least 20%, 50%, 80% or moreof peak activity. Typically the pH is from 3 to 11, such as 5 to 9 or 6to 8, preferably 7 to 7.8 or 7.4. Typically the temperature is 10 to 90°C., such as 25 to 75° C. or 30 to 60° C.

In the process different monooxygenases may be present. Typically eachof these will be able to oxidise different substrates, and thus using amixture of monooxygenases will enable a wider range of substrates to beoxidised.

In one embodiment the process is carried out in the presence of asubstance able to remove hydrogen peroxide by-product (e.g. a catalase).

In one embodiment the process is carried out in the presence of theenzyme, substrate and an oxygen atom donor, such as hydrogen peroxide ort-butylhydroperoxide, for example using the peroxide shunt.

In one embodiment in the process the (a), (b) and (c) together aretypically in a substantially isolated form and/or a substantiallypurified form, in which case together they will generally comprise atleast 90%, e.g., at least 95%, 98% or 99% of the protein in thepreparation.

The process may be carried out inside or outside a cell. The cell istypically in culture, at a locus, in vivo or in planta (these aspectsare discussed below).

The process is typically carried out at a locus such as in land (e.g insoil) or in water (e.g, fresh water or sea water). When it carried outin culture the culture typically comprises different types of cells ofthe invention, for example expressing different monooxygenases of theinvention. Generally such cells are cultured in the presence ofassimible carbon and nitrogen sources.

Typically the cell in which the process is carried out is one in whichthe monooxygenase does not naturally occur. In another embodiment themonooxygenase is expressed in a cell in which it does naturally occur,but at higher levels than naturally occurring levels. The cell mayproduce 1, 2, 3, 4 or more different monooxygenases of the invention.These monoxygenases may be capable of oxidizing different halo aromaticcompounds. Typically the cell also expresses any of the reductasesand/or redoxins discussed above.

The cell is typically produced by introducing into a cell (i.e.transforming the cell with) a vector comprising a polynucleotide thatencodes the monooxygenase. The vector may integrate into the genome ofthe cell or remain extrachromosomal. The cell may develop into theanimal or plant discussed below. Typically the coding sequence of thepolynucleotide is operably linked to a control sequence which is capableof providing for the expression of the coding sequence by the host cell.The control sequence is generally a promoter, typically of the cell inwhich the monooxygenase expressed.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences.

The vector is typically a transposon, plasmid, virus or phage vector. Ittypically comprises an origin of replication. It typically comprises oneor more selectable marker genes, for example an ampicillin resistancegene in the case of a bacterial plasmid. The vector is typicallyintroduced into host cells using conventional techniques includingcalcium phosphate precipitation, DEAE-dextran transfection, orelectroporation.

Components (b) and (c) may be expressed in the cell in a similar manner.Typically (a), (b) and (c) are expressed from the same vector, or may beexpressed from different vectors. They may be expressed as threedifferent polypeptides. Alternatively they may be expressed in the formof fusion proteins. The cell typically expresses more than one type ofmonooxygenase.

In one embodiment the three genes encoding the three proteins of theP450_(cam) system, i.e. camA, camB, and camC are placed in the mobileregions of standard transposon vectors and incorporated into the genomeof Pseudomonas and flavobacteria. Alternatively plasmid vectors forexpressing these genes may used, in which case the P450_(cam) genecluster will be extra-chromosomal.

The cell may be prokaryotic or eukaryotic and is generally any of thecells or of any of the organisms mentioned herein. Preferred cells arePseudomanas, flavobacteria or fungi cells (e.g. Aspergillus). In oneembodiment the cell is one which in its naturally occurring form is ableto oxidise any of the substrates mentioned herein. Typically the cell isin a substantially isolated form and/or substantially purified form, inwhich case it will generally comprise at least 90%, e.g. at least 95%,98% or 99% of the cells or dry mass of the preparation.

The invention provides a transgenic animal or plant whose cells are anyof the cells of the invention. The animal or plant is transgenic for themonooxygenase gene and typically also an appropriate electron transferreductase and/or redoxin gene. They may be homozygote or heterozygotefor such genes, which are typically transiently introduced into thecells, or stably integrated (e.g. in the genome). The animal istypically a worm (e.g earthworm) or nematode. The plant or animal may beobtained by transforming an appropriate cell (e.g. embryo stem cell,callus or germ cell), fertilising the cell if required, allowing thecell to develop into the animal or plant and breeding the animal orplant true if required. The animal or plant may be obtained by sexual orasexual (e.g cloning) propagation of an animal or plant of the inventionor of the F1 organism (or any generation removed from the F1, or thechimera that develops from the transformed cell).

As discussed above the process may be carried out at a locus. Thus theinvention also provides a method of treating a locus contaminated with ahalo aromatic compound comprising contacting the locus with amonooxygenase, cell, animal or plant of the invention. These organismsare then typically allowed to oxidise the halo aromatic compound. In oneembodiment the organisms used to treat the locus are native to thelocus. Thus they may be obtained from the locus (e.g. aftercontamination), transformed/transfected (as discussed above) to expressthe monooxygenase (and optionally an appropriate electron transferreductase and/or redoxin.

In one embodiment the locus is treated with more than one type oforganism of the invention, e.g. with 2, 3, 4, or more types whichexpress different monooxygenases which oxidise different halo aromaticcompounds. In one embodiment such a collection of organisms between themis able to oxidise all halobenzenes, e.g. all chlorobenzenes.

The organisms (e.g. in the form of the collection) may carry out theprocess of the invention in a bioreactor (e.g. in which they are presentin immobilised form). Thus the water or soil to be treated may be passedthrough such a bioreactor. Soil may be washed with water augmented withsurfactants or ethanol and then introduced into the bioreactor.

The invention also provides a process for selecting a mutant of amonooxygenase for its ability to oxidise any of the substrates mentionedherein, which process comprises screening a library of said mutants fortheir oxidation effect on the substrate. Thus typically the substrate isprovided to the library and mutants are selected based on their abilityto oxidise the substrate, for example at a particular rate or underparticular conditions. The mutant may be selected based on its abilityto oxidise the substrate to a particular oxidation product.

Typically the library will be in the form of cells which comprise themutant enzymes. Generally each cell will express only one particularmutant enzyme. The library typically comprises at least 500 mutants,such as at least 1,000 or 5,000 mutants, preferably at least 10,000different mutants.

The library typically comprises a random population of mutants. Thelibrary may undergo one or more rounds of selection whilst beingproduced and therefore may not comprise a random population.

The library is typically produced by contacting any of the cellsdiscussed herein which expresses the monooxygenase with a mutagen and/orwhen the cell is a mutator cell culturing the cell in conditions inwhich mutants are produced. The mutagen may be contacted with the cellprior to or during culturing of the cell. Thus the mutagen may bepresent during replication of the cell or replication of the genome ofthe cell.

The mutagen generally causes random mutations in the polynucleotidesequence which encodes (a). The mutagen is typically a chemical mutagen,such as nitrosomethyguanidine, methyl- or ethylmethane sulphonic acid,nitrite, hydroxylamine, DNA base analogues, and acridine dyes, such asproflavin. It is typically electromagnetic radiation, such asultra-violet radiation at 260 nm (absorption maximum of DNA) and X-rays.It is typically ionising radiation.

A mutator cell is generally deficient in one or more of the primary DNArepair pathways (such as E. Coli pathways mutS, mutD or mutT, or theirequivalents in another organism), and thus has a high mutation rate.Simply culturing such cell leads to the DNA encoding (a) to becomemutated. The cell may be of E. Coli XL1 Red mutator strain.

The mutant selected from the library may be used in any aspect of theinvention, thus it may be used to oxidise a substrate in the process ofthe invention or may be expressed in the cell, animal or plant of theinvention. It may be used in the method of treating a locus.

The invention is also illustrated by the Examples:

EXAMPLE 1 Expression of Mutants for in vitro Work

The P450_(cam) enzymes were expressed using the vector pRH1091 (Baldwin,J. E., Blackburn, J. M., Heath, R. J., and Sutherland, J. D. Bioorg.Med. Chem. Letts., 1992, 2, 663-668.) which utilised the trc promoter (afusion of the trp and lac promoters). This vector incorporates a strongribosome binding site (RBS) and the gene to be expressed is cloned usingan Nde I site on the 5′ end of the gene. We used Hind III as the cloningsite at the 3′ end of the camC gene. The procedure for proteinexpression is as follows: Cells are grown at 30° C. until theOD_(600 nm) reaches 1.0-1.2, the temperature is increased to 37° C. andcamphor added as a 1 M stock in ethanol to a final concetration of 1 mM.The culture is allowed to incubate at 37° C. for another 6 hours. TheP450_(cam) protein is expressed to high levels in the cytoplasm and thecells take on a red to orange-red colour.

We have also prepared a variant of pRH1091 (by PCR) which has a extraXba I site between the RBS and the Nde I site. This is important becauseNde I is not unique in M13, and this restriction site is also present inthe reductase gene as well as the backbone of the pGLW11 vector used forthe in vivo system. Xba I is unique in the polylinker region of M13, butabsent in the genes of all three proteins in the P450_(cam) system andin the expression vectors. It therefore allows the camC gene to be movedbetween the mutagenic and expression vectors.

How the Mutants were Made

Oligonucleotide-directed site-specific mutagenesis was carried out bythe Kunkel method (Kunkel, T. A. Proc. Natl. Acad. Sci. USA 1985, 82,488-492) using the Bio-Rad Mutagen kit. The recommended procedure issummarised as follows. An M13 mp19 subclone of the camC gene encodingP450_(cam) (SEQ ID NO: 1) was propagated in the E. coli strain CJ236.This strain has the dut⁺ung⁺ phenotype and thus will tolerate theinclusion of uracil in place of thymine in DNA molecules. After threecycles of infection, uracil-containing single stranded (USS) M13 DNA wasreadily isolated by phenol extraction of mature M13 phage particlesexcreted into the growth medium. The mutagenic oligonucloetide (oroligonucleotides) were phosphorylated with T4 polynucleotide kinase andthen annealed to the USS template. The four nucleotides, DNA polymerase,DNA ligase, ATP and other chemical components were added and the secondstrand was synthesised in vitro. The double stranded form thus obtainedwas transformed into the dut+ ung+ E. coli strain MV1190, which shoulddegrade the uracil-containing template strand and propagate the mutantstrand synthesised in vitro. Plaques were picked and phages of possiblemutants grown in E. coli strains MV1190 or TG1. The single-stranded DNAfrom these were sequenced to determine whether the mutagenesis, reactionwas successful. The mutagenic efficiency was 50-80%.

The mutant camC gene is excised from the M13 subclone by restrictiondigest with Nde I and Hind III, and the fragment of appropriate size isligated to the backbone of the expression vector prepared by a similarNde I/Hind III digest.

Multiple mutants were prepared either by further mutagenesis, also bythe Kunkel method, or where the location of the sites in the sequencepermits, simple cloning steps. There are two unique restriction siteswithin the camC gene which are absent from the expression vector. One isSph I which spans residues 121-123, and the other is Sal I which spansresidues 338 and 339. Therefore, all mutations at, for example, residues87, 96, 98, and 101 are readily combined with mutations at higher numberresidues by ligating appropriate fragments from restriction digests ofmutant camC genes with Nde I/Sph I and Sph I/Hind III and the backbonefragment from a Nde I/Sph I digest of the expression vector. Mutationsat, for example, 395 and 396 can be similarly incorporated by digests inwhich Sph I is replaced with Sal I.

The rationale for introducing the unique Xba I site is now clear: manymutants with multiple mutations were prepared by the cloning procedureabove. Without the Xba I site it would be impossible to clone the genefor these multiple mutants from the expression vector back into M13 forfurther rounds of mutagenesis. Of course these problems could beovercome by doing mutagenesis by PCR, for example.

EXAMPLE 2

Substrate oxidation protocol: in vitro reactions Component Finalconcentration P450_(cam) enzyme 1 μM Putidaredoxin 10 μM Putidaredoxinreductase 1 μM Bovine liver catalase 20 μg/ml KCl 200 mM SubstrateTypically 1 mM NADH 250-400 μM

50 mM Tris-HCl buffer pH 7.4 is added to make up the volume.

Temperature controlled at 30° C., optional.

The NADH turnover rate could be determined by monitoring the absorbanceat 340 nm with time.

Catalase does not catalyse the substrate oxidation reactions but ratherit is present to remove any hydrogen peroxide by-product which couldotherwise denature the P450_(cam).

The method can be increased in scale to, for example, 20 ml totalincubation volume to allow purification of sufficient products by HPLCfor spectroscopic characterisation. Fresh substrate (1 mM) and NADH (1-2mM) are added periodically, such as every 20 minutes in a total rectiontime of, typically, 3 hours.

EXAMPLE 3 The in vivo System

The in vivo systems were expressed using the vector pGLW11, a derivativeof the plasmid pKK223 (Brosius, J. and Holy, A. Proc. Natl. Acad. Sci.USA, 1984, 81, 6929-6933). Expression is directed by the tac promoterand the vector incorporates a gene conferring resistance to theantibiotic ampicillin.

Two systems were constructed. The first one expressed the electrontransfer proteins putidaredoxin reductase (camA gene) and putidaredoxin(camB gene) as a fusion protein with a seven amino acid peptide linker,and the P450_(cam) enzyme (camC gene) was expressed by the same vectorbut it was not fused to the electron proteins. The second systemexpressed the three proteins as separate entities in the E. Coli host.Both systems were catalytically competent for substrate oxidation invivo.

The general strategy was as follows. The genes for the three proteinswere cloned using Eco RI and Hind III as flanking sites, with Eco RI atthe 5′ end. For both in vivo systems there are restriction sites betweenthe genes, including between the reductase and redoxin genes in thefusion construct. These restriction sites were introduced by PCR, asdetailed below. The first task, however, was to carry out a silentmutation to remove the Hind III site within the camA gene for thereductase. The AAGCTT Hind III recognition sequence in the came gene waschanged to AAGCCT, which is a silent mutation because GCT and GCC bothencode alanine. The gene was completely sequenced to ensure that therewere no spurious mutations.

1. The Fusion Protein System

1.a Manipulation of the camA Gene by PCR

For the camA gene the primer below (SEQ ID NO: 5) was used at the 5′ endof the gene to introduce the Eco RI cloning site and to change the firstcodon from GTG to the strong start codon ATG.

5′-GAG ATT AAG AAT TCA TAA ACA CAT GGG AGT GCG TGC CAT ATG AAC GCA AAC           Eco RI    RBS      |→camA

At the 3′ end of cam the primer (nucleotide sequence is SEQ ID NO: 7)was designed such that 15 bases are complementary to nucleotide sequenceof the last five amino acid residues of camA. The stop codon immediatelyafter the GCC codon for the last amino acid was removed, and then partof a seven amino acid linker (Thr Asp Gly Gly Ser Ser Ser; SEQ ID NO: 6)which contained a Bam HI cloning site (GGATCC=Gly Ser) was introduced.The coding sequence was thus (amino acid sequence is SEQ ID NO:8):

       5′-GAA CTG AGT AGT GCC ACT GAC GGA GGA TCC TCA TCG-3′               camA     →Thr Asp Gly Gly Ser                                         |Bam HI|

The primer sequence shown below (SEQ ID NO: 9) is the reverse complementused for PCR:

5′-CGA TGA GGA TCC TCC GTC AGT GGC ACT ACT CAG TTC-3′

1.b Manipulations of the camB Gene by PCR

For the camB gene the primer at the 5′ end (nucleotide sequence is SEQID NO: 10; amino acid sequence is SEQ ID NO: 11) incorporated the secondhalf of the peptide linker between the reductase and redoxin proteins,and the restriction site Bam HI for joining the two amplified genestogether.

       5′-TCA TCG GGA TCC TCA TCG ATG TCT AAA GTA GTG TAT-3′               Gly Ser Ser Ser|→ CamB                |BamHI|         Start

At the 3′ end of camB the primer incorporates 12 nucleotidescomplementary to the end of camB followed by the stop codon TAA, a 6nucleotide spacer before the GGAG ribosome binding site. Xba I and HindIII sites were then added to allow cloning of the camC gene whenrequired. The sequence of the coding strand was therefore (SEQ ID NO:12):

5′-CCC GAT AGG CAA TGG TAA TCA TCG GGAG TCT AGA GCA TCG AAG CTT TCATCG-3′                CamB →|stop    RBS Xba I Hind III

The primer shown below (SEQ ID NO: 13) is the reverse complement usedfor PCR:

5′-CGA TGA AAG CTT CGA TGC TCT AGA CTCC CGA TGA TTA CCA TTG CCT ATCGGG-3′

1.c Preparation of the Full Fusion Construct

The camA and camB genes were amplified by the PCR using the primersdescribed above. The new camA was digested with Eco RI and Bam HI, whilethe new CamB was digested with Bam HI and Hind III. The pGLW11expression vector was digested with Eco RI and Hind III. All three werepurified by agarose gel electrophoresis and the three gel slicescontaining the separate fragments were excised from the gel and ligatedtogether, and then transformed into E. Coli DH5α. Successful ligation ofall the fragments were confirmed by a series of restriction digestionexperiments, especially the presence of the new and unique Xba I site.The entire sequence of the insert from the Eco RI site to the Hind IIIsite was determined to ensure that all the sequences were correct.

The new plasmid, named pSGB^(F), was transformed into E. Coli andexpression of the reductase and redoxin proteins was induced by IPTG.When a purified P450_(cam) enzyme was added to the cell-free extract,substrate oxidation was observed for a variety of substrates.

When the camC gene is cloned into the pSGB^(F) plasmid using the Xba Iand Hind III restriction sites, the new recombinant plasmid thusgenerated expresses the reductase and redoxin as a fusion protein andthe P450_(cam) enzyme as a operate entity both from the same mRNAmolecule. This in vivo system is catalytically competent for terpeneoxidation in whole cells.

2. The in vivo System with the Protein Expressed Separately

2.a The Basic Strategy

The starting point of the preparation of this in vivo system was therecombinant plasmid used to express the camA gene for putidaredoxinreductase. The camA gene was cloned into the pGLW11 plasmid using theEco RI and Bam HI restriction sites, with Eco RI being at the 5′ end ofthe gene. Conveniently the polylinker region of the pGLW11 vector has aHind III site downstream of the Bam HI site. The camB gene was thereforemanipulated by PCR such that it can be cloned into pGLW11 using the BamHI and Hind III sites. This new plasmid expresses the reductase andredoxin as separate proteins.

The camB gene was cloned into pUC118 by the Bam HI and Hind III cloningsites to express putidaredoxin for our general in vitro substrateoxidation work. Therefore, the PCR primer at the 3′ end of the camB genewas designed to introduce a ribosome binding site and the Xba Irestriction site upstream of the Hind III site so that the camC gene canbe inserted downstream of camB using the Xba I and Hind III sites.Therefore the three genes were cloned without fusion in the pGLW11expression vector and arranged in the order 5′-camA-camB-camC-3′, andeach gene has its own RBS to initiate protein synthesis.

2.b Manipulations of the camB Gene

We used the internal and unique restriction site Mlu I (recognitionsequence ACGCGT) within the camB gene as the starting point so that thePCR product has a different size from the PCR template fragment. Theprimers were as follows:

5′-TCA TCG ACG CGT CGC GAA CTG CTG-3′ (SEQ ID NO: 14)

where the Mlu I site is in bold.

The desired coding sequence at the 3′ end of the camB gene (SEQ IDNO:15) was:

   5′-CCC GAT AGG CAA TGG TAA GTA GGT GAA TAT CTA ATC CCC ATC TAT GCGCGA GTG GAG TCT AGA GTT CGA-3′        camB   →|stop                                                  RBS  XbaI

After the stop codon there is a 35 base spacer before the RBS which isused to initiate the synthesis of the P450_(cam) enzyme. The Xba Icloning site is located within the spacer between the RBS and the startcodon (not in this primer) of the camC gene. The PCR primer used was thereverse complement of the sequence above. The PCR was carried out andthe amplified fragment of the appropriate size was purified by agarosegel electrophoresis and the gel slice excised.

One extra step was necessary to complete the construction of the newplasmid. The plasmid for the fusion protein in vivo system was digestedwith Mlu I and Hind III restriction enzymes, purified by agarose gelelectrophoresis, and the gel slice for the small camB fragment excise.The pUC118 plasmid for camB expression was similarly digested, and thegel slice for the backbone was excised. By ligating the two fragmentstogether we prepared a new pUC118-based plasmid which had an Xba I sitefollowed by an Hind III site downstream of the stop codon of camB. Thisnew plasmid was digested with the Mlu I and Xba I enzymes and thebackbone was ligated with the new camB fragment described above togenerate a plasmid with the following arrangement of the key components:

..lac Promoter..Bam HI..camB gene..spacer..RBS..Xba I..Hind III..

2.c Preparation of the in vivo System Plasmid

Once the modified camB with the Xba I and Hind III restriction sites andappropriate spacers were prepared, the in vivo system was constructed bycloning this into the pGLW11-based plasmid used to express the camA gene(reductase protein) using the Bam HI and Hind III sites. The new in vivosystem vector has the following arrangement of the key components:

..tac Promoter..Eco IRI..RBS..camA gene..spacer..Bam HI..RBS..camBgene..spacer..RBS..Xba I..Hind III..

This new plasmid, named pSGB⁺, was transformed into E. Coli andexpression of the reductase and redoxin proteins was induced by IPTG.When a purified P450_(cam) enzyme was added to the cell-free extract,substrate oxidation was observed for a variety of substrates.

When the camC gene is cloned into this pSGC⁺ plasmid using the Xba I andHind III restriction sites, the new recombinant plasmid thus generatedwill express the three proteins separately, each under the direction ofits own RBS but from the same mRNA molecule. Thus constitutes the invivo system used in the vast majority of our terpene oxidation work.

3. Introduction of an Xba I Site Into pRH1091

This is the final step to enable the camC gene to be cloned into the invivo systems by the two cloning sites XbaI and Hind III. The Xba I sitewas added by PCR of the entire pRH1091 plasmid using two primers. Thepresence of these two sites will also enable cloning of the camC geneinto M13 since both Xba I and Hind III are unique in camC and M13.

The primers shown below maintain the Hind III cloning site AAGCTT:

5′-TCA TCG AAG CTT GGC TGT TTT-3′ (SEQ ID NO:16)       Hind III|→ vector

At the other end the coding sequence desired was (SEQ ID NO: 17):

5′-ACA ATT TCA CAC AGGA TCT AGA C CAT ATG TCA TCG AAG CTT TCA TCG-3′   Vector →|RBS XbaI  NdeI    Hind III

This sequence maintained the Nde I and Hind III sites but the new Xba Isite was introduced upstream of the Nde I site. The PCR primer used wasthe reverse complement of the desired sequence (SEQ ID NO: 18):

5′-CGA TGA AAG CTT CGA TGA CAT ATG GTC T AGA TCCT GTG TGA AAT TGT-3′

The PCR product was then purified by agarose gel electrophoresis,digested with Hind III and circularised with T4 DNA ligase. Success ofthe PCR method was indicated by the presence of a new and unique Xba Isite in plasmid DNA isolated from transformants.

4. Cloning of camC Into the in vivo Systems

All existing camC mutants were cut out of pRH1091-based expressionplastids with Nde I and Hind III. The new vector is similarly cut withthe same restriction enzymes and the camC gene cloned into this plasmidwith T4 DNA ligase. This DNA is transformed into E. Coli JM109 whichthen may be grown to express P450_(cam).

The camC gene is excised from the new vector using Xba I and Hind IIIrestriction enzymes and cloned into either the in vivo vector systems orM13mp19 for mutagenesis.

5. In vivo Expression and Substrate Turnover

For protein expression, cells are grown in LBamp medium (tryptone 10g/liter, yeast extract 5 g/liter, NaCl 10 g/liter, 50 μg/ml ampicillin)at 30° C. until the OD_(600 nm) reaches 1.0-1.2. IPTG(isopropyl-β-D-thiogalactopyranoside) was added to a final concentrationof 1 μM (from a 1 M stock in H₂O) and the culture was incubated at 30°C. overnight.

For simple screening the substrate can be added to culture and theincubation continued. However, due to impurities from the culture mediathe cells were generally washed twice with 0.5 vol. of buffer P, (KH₂PO₄6.4 g, K₂HPO₄.3H₂O 25.8 g, H₂O to 4 liters, pH 7.4) and resuspended in0.25 vol. oxygen saturated buffer P containing 24 mM glucose. Substratewas added to 1 mM and the incubation continued at 30° C. The reactionwas allowed to run for 24 hours with periodic additions of substrate andglucose.

EXAMPLE 4 The Oxidation of Halo Aromatic Compounds

The oxidation of halo aromatic compounds 2,3,6- 3,4,6- Coupling ProductTrichloro- Trichloro- Efficiency formation Mutant phenol phenol (%) rate(min⁻¹) Y96F 75 25 18  22 Y96A 77 23 14  33 Y96H 54 46 3 1 F87L-Y96F 4258 4 8 F87A-Y96F 52 48 2 3 F87A-Y96-F-V247A 43 57 4 7 2,3- 3,4- CouplingProduct Dichloro- Dichloro- Efficiency formation Mutant phenol phenol(%) rate (min⁻¹) Y96A 94  6 6 19 Y96F 91  9 4 8 Y96A-V247L 94  6 7 20Y96L-V247A 90 10 2 0.7 F87L-96F 96  4 3 5 C334A 95  5 2 0.5

All mutants have C334A. Coupling efficiency is the percentage of NADHconsumed which was utilised for product formation, i.e. a percentage ofthe theoretical maximum efficiency. The product formation rates aregiven in (nmol product) (nmol P450_(cam))⁻¹(min)⁻¹. The relative amountof product formed in each case is shown. 1,3- and 1,4-dichlorobenzene,1,2,3- and 1,3,5-trichlorobenzene, 1,2,4,5- and1,2,3,5-tetrachlorobenzene, and 2,3,4,5,6- and2,2′,4,5,5′-pentachlorobiphenyl were also found to be oxidised.

Wild-type and mutant P450_(cam) enzymes were tested for their ability tooxidise 3,3′-dichlorobiphenyl and 2,2′,4,5,5′-pentachlorobiphenyl.Results are shown in terms of NADH turnover. Rates are given as nanomolNADH consumed per nanomol P450_(cam) enzyme per minute.

3,3′- 2,2′,4,5,5′- P450_(cam) enzyme dichlorobiphenylpentachlorobiphenyl Wild-type 0.4 not detected Y96F 15  1 F87A-Y96F 845165 F87L-Y96F 174  13 F87W-Y96F 4  3 F87A-Y96F-V247A 112  12 Y96A-V247L84  37 F87A-Y96F-L244A 669 321 P87A-Y97F-L244A-V247A 173 214

The first product, 4-hydroxy-3,3′-dichlorobiphenyl was identified by thecharacteristic coupling patterns expected in the ¹H NMR spectrum and bymass spectroscopy. The further oxidation product,4,4′-dihydroxy-3,3′-dichlorobiphenyl was identified by co-elution withan authentic sample, and by UV-vis and mass spectroscopy. This productdid not constitute more than ca. 10% of the total products in any of themutants tested.

For the second substrate product was established as4′-hydroxy-2,2′,4,5,5′-pentachlorobiphenyl by the observation of theparent ion in the mass spectrum, and by comparison with literature ¹HNMR data

P450_(cam) Mutants

All mutants optionally contain the base mutation C334A.

Single mutants: Y96A, Y96F, Y96L, Y96W. Double mutants: Y96A-F87AY96F-F87A Y96F-V295A Y96L-F87A Y96L-A296L Y96A-F87L Y96F-F87I Y96F-V295LY96L-F87L Y96L-A296F Y96A-F87W Y96F-F87L Y96F-V295I Y96L-F98W Y96L-V396AY96A-F98W Y96F-F87W Y96F-A296L Y96L-T101L Y96L-V396L Y96A-L244AY96F-F98W Y96F-A296F Y96L-T101F Y96L-V396F Y96A-V247A Y96F-T101LY96F-I395F Y96L-L244A Y96L-V396W Y96A-V247L Y96F-T101F Y96F-I395GY96L-L244F Y96A-I395F Y96F-T185A Y96F-V396A Y96L-V247A Y96A-I395GY96F-T185F Y96F-V396L Y96L-V247L Y96W-F87W Y96F-T185L Y96F-V396FY96L-V247F Y96W-F98W Y96F-L244A Y96F-V396W Y96L-V247W Y96W-L244AY96F-V247A Y96L-G248L Y96W-V247A Y96F-V247L Y96L-V295L Y96W-V396AY96F-G248L Y96L-V295F Triple Mutants: Y96A-F87A-L244A Y96L-V247A-V396LY96F-F87W-V247A Y96A-F87A-V247A Y96L-V247A-V396F Y96F-F87W-V247LY96A-F87L-L244A Y96L-V247A-V396W Y96F-F87W-V247F Y96A-F87L-V247AY96L-V247F-V396A Y96F-F87W-V295L Y96A-L244A-V247A Y96F-F87W-A296LY96F-F87A-L244A Y96F-F87W-V396A Y96L-F87A-L244A Y96F-F87A-V247AY96F-F87W-V396L Y96L-F87A-V247A Y96F-F87A-V247L Y96F-V247F-V396AY96L-F87L-L244A Y96F-F87A-I395F Y96F-L244A-V396L Y96L-F87L-V247AY96F-F87A-I395G Y96F-L244A-V396F Y96L-V247A-I395F Y96F-F87L-V247AY96F-L244A-V396W Y96L-V247L-I395F Y95F-F87L-V247L Y96F-L244F-V396AY96L-V247L-I395G Y96F-F87L-I395F Y96F-V247A-V396L Y96L-L244A-V396LY96F-F87W-T185A Y96F-V247A-V396F Y96L-L244A-V396F Y96F-F87W-T185FY96F-V247A-V396W Y96L-L244A-V396W Y96F-F87W-T185L Y96L-L244F-V396AY96F-F87W-L244F Y96W-F87W-F98W Four mutations: Five mutations:Y96A-F87A-L244A-V247A Y96F-F87W-T185L-V247L-V295L Y96A-F87L-L244A-V247AY96F-F87W-T185L-V247L-V396A Y96L-F87A-L244A-V247AY96F-F87W-T185L-V247L-V396L Y96L-F87L-L244A-V247A Y96F-F87W-L244A-V295LY96F-F87W-L244F-V396A Y96F-F87W-L244A-A296L Y96F-F87W-V247A-V396LY96F-F87W-V247A-V396F Y96F-F87W-V247L-V295A Y96F-F87W-V247L-V396AY96F-F87W-V247F-V396A Y96F-F87W-V247A-I395F Y96F-F87W-V247L-I395G

18 1 1242 DNA Pseudomonas putida 1 acgactgaaa ccatacaaag caacgccaatcttgcccctc tgccacccca tgtgccagag 60 cacctggtat tcgacttcga catgtacaatccgtcgaatc tgtctgccgg cgtgcaggag 120 gcctgggcag ttctgcaaga atcaaacgtaccggatctgg tgtggactcg ctgcaacggc 180 ggacactgga tcgccactcg cggccaactgatccgtgagg cctatgaaga ttaccgccac 240 ttttccagcg agtgcccgtt catccctcgtgaagccggcg aagcctacga cttcattccc 300 acctcgatgg atccgcccga gcagcgccagtttcgtgcgc tggccaacca agtggttggc 360 atgccggtgg tggataagct ggagaaccggatccaggagc tggcctgctc gctgatcgag 420 agcctgcgcc cgcaaggaca gtgcaacttcaccgaggact acgccgaacc cttcccgata 480 cgcatcttca tgctgctcgc aggtctaccggaagaagata tcccgcactt gaaataccta 540 acggatcaga tgacccgtcc ggatggcagcatgaccttcg cagaggccaa ggaggcgctc 600 tacgactatc tgataccgat catcgagcaacgcaggcaga agccgggaac cgacgctatc 660 agcatcgttg ccaacggcca ggtcaatgggcgaccgatca ccagtgacga agccaagagg 720 atgtgtggcc tgttactggt cggcggcctggatacggtgg tcaatttcct cagcttcagc 780 atggagttcc tggccaaaag cccggagcatcgccaggagc tgatcgagcg tcccgagcgt 840 attccagccg cttgcgagga actactccggcgcttctcgc tggttgccga tggccgcatc 900 ctcacctccg attacgagtt tcatggcgtgcaactgaaga aaggtgacca gatcctgcta 960 ccgcagatgc tgtctggcct ggatgagcgcgaaaacgcct gcccgatgca cgtcgacttc 1020 agtcgccaaa aggtttcaca caccacctttggccacggca gccatctgtg ccttggccag 1080 cacctggccc gccgggaaat catcgtcaccctcaaggaat ggctgaccag gattcctgac 1140 ttctccattg ccccgggtgc ccagattcagcacaagagcg gcatcgtcag cggcgtgcag 1200 gcactccctc tggtctggga tccggcgactaccaaagcgg ta 1242 2 414 PRT Pseudomonas putida 2 Thr Thr Glu Thr IleGln Ser Asn Ala Asn Leu Ala Pro Leu Pro Pro 1 5 10 15 His Val Pro GluHis Leu Val Phe Asp Phe Asp Met Tyr Asn Pro Ser 20 25 30 Asn Leu Ser AlaGly Val Gln Glu Ala Trp Ala Val Leu Gln Glu Ser 35 40 45 Asn Val Pro AspLeu Val Trp Thr Arg Cys Asn Gly Gly His Trp Ile 50 55 60 Ala Thr Arg GlyGln Leu Ile Arg Glu Ala Tyr Glu Asp Tyr Arg His 65 70 75 80 Phe Ser SerGlu Cys Pro Phe Ile Pro Arg Glu Ala Gly Glu Ala Tyr 85 90 95 Asp Phe IlePro Thr Ser Met Asp Pro Pro Glu Gln Arg Gln Phe Arg 100 105 110 Ala LeuAla Asn Gln Val Val Gly Met Pro Val Val Asp Lys Leu Glu 115 120 125 AsnArg Ile Gln Glu Leu Ala Cys Ser Leu Ile Glu Ser Leu Arg Pro 130 135 140Gln Gly Gln Cys Asn Phe Thr Glu Asp Tyr Ala Glu Pro Phe Pro Ile 145 150155 160 Arg Ile Phe Met Leu Leu Ala Gly Leu Pro Glu Glu Asp Ile Pro His165 170 175 Leu Lys Tyr Leu Thr Asp Gln Met Thr Arg Pro Asp Gly Ser MetThr 180 185 190 Phe Ala Glu Ala Lys Glu Ala Leu Tyr Asp Tyr Leu Ile ProIle Ile 195 200 205 Glu Gln Arg Arg Gln Lys Pro Gly Thr Asp Ala Ile SerIle Val Ala 210 215 220 Asn Gly Gln Val Asn Gly Arg Pro Ile Thr Ser AspGlu Ala Lys Arg 225 230 235 240 Met Cys Gly Leu Leu Leu Val Gly Gly LeuAsp Thr Val Val Asn Phe 245 250 255 Leu Ser Phe Ser Met Glu Phe Leu AlaLys Ser Pro Glu His Arg Gln 260 265 270 Glu Leu Ile Glu Arg Pro Glu ArgIle Pro Ala Ala Cys Glu Glu Leu 275 280 285 Leu Arg Arg Phe Ser Leu ValAla Asp Gly Arg Ile Leu Thr Ser Asp 290 295 300 Tyr Glu Phe His Gly ValGln Leu Lys Lys Gly Asp Gln Ile Leu Leu 305 310 315 320 Pro Gln Met LeuSer Gly Leu Asp Glu Arg Glu Asn Ala Cys Pro Met 325 330 335 His Val AspPhe Ser Arg Gln Lys Val Ser His Thr Thr Phe Gly His 340 345 350 Gly SerHis Leu Cys Leu Gly Gln His Leu Ala Arg Arg Glu Ile Ile 355 360 365 ValThr Leu Lys Glu Trp Leu Thr Arg Ile Pro Asp Phe Ser Ile Ala 370 375 380Pro Gly Ala Gln Ile Gln His Lys Ser Gly Ile Val Ser Gly Val Gln 385 390395 400 Ala Leu Pro Leu Val Trp Asp Pro Ala Thr Thr Lys Ala Val 405 4103 3150 DNA Bacillus megaterium 3 atgacaatta aagaaatgcc tcagccaaaaacgtttggag agcttaaaaa tttaccgtta 60 ttaaacacag ataaaccggt tcaagctttgatgaaaattg cggatgaatt aggagaaatc 120 tttaaattcg aggcgcctgg tcgtgtaacgcgctacttat caagtcagcg tctaattaaa 180 gaagcatgcg atgaatcacg ctttgataaaaacttaagtc aagcgcttaa atttgtacgt 240 gattttgcag gagacgggtt atttacaagctggacgcatg aaaaaaattg gaaaaaagcg 300 cataatatct tacttccaag cttcagtcagcaggcaatga aaggctatca tgcgatgatg 360 gtcgatatcg ccgtgcagct tgttcaaaagtgggagcgtc taaatgcaga tgagcatatt 420 gaagtaccgg aagacatgac acgtttaacgcttgatacaa ttggtctttg cggctttaac 480 tatcgcttta acagctttta ccgagatcagcctcatccat ttattacaag tatggtccgt 540 gcactggatg aagcaatgaa caagctgcagcgagcaaatc cagacgaccc agcttatgat 600 gaaaacaagc gccagtttca agaagatatcaaggtgatga acgacctagt agataaaatt 660 attgcagatc gcaaagcaag cggtgaacaaagcgatgatt tattaacgca tatgctaaac 720 ggaaaagatc cagaaacggg tgagccgcttgatgacgaga acattcgcta tcaaattatt 780 acattcttaa ttgcgggaca cgaaacaacaagtggtcttt tatcatttgc gctgtatttc 840 ttagtgaaaa atccacatgt attacaaaaagcagcagaag aagcagcacg agttctagta 900 gatcctgctc caagctacaa acaagtcaaacagcttaaat atgtcggcat ggtcttaaac 960 gaagcgctgc gcttatggcc aactgctcctgcgttttccc tatatgcaaa agaagatacg 1020 gtgcttggag gagaatatcc tttagaaaaaggcgacgaac taatggttct gattcctcag 1080 cttcaccgtg ataaaacaat ttggggagacgatgtggaag agttccgtcc agagcgtttt 1140 gaaaatccaa gtgcgattcc gcagcatgcgtttaaaccgt ttggaaacgg tcagcgtgcg 1200 tgtatcggtc agcagttcgc tcttcatgaagcaacgctgg tacttggtat gatgctaaaa 1260 cactttgact ttgaagatca tacaaactacgagctggata ttaaagaaac tttaacgtta 1320 aaacctgaag gctttgtggt aaaagcaaaatcgaaaaaaa ttccgcttgg cggtattcct 1380 tcacctagca ctgaacagtc tgccaaaaaagcacgcaaaa aggcagaaaa cgctcataat 1440 acgccgctgc ttgtgctata cggttcaaatatgggaacag ctgaaggaac ggcgcgtgat 1500 ttagcagata ttgcaatgag caaaggatttgcaccgcagg tcgcaacgct tgattcacac 1560 gccggaaatc ttccgcgcga aggagctgtattaattgtaa cggcgtctta taacggtcat 1620 ccgcctgata acgcaaagca atttgtcgactggttagacc aagcgtctgc tgatgaagta 1680 aaaggcgttc gctactccgt atttggatgcggcgataaaa actgggctac tacgtatcaa 1740 aaagtgcctg cttttatcga tgaaacgcttgccgctaaag gggcagaaaa catcgctgac 1800 cgcggtgaag cagatgcaag cgacgactttgaaggcacat atgaagaatg gcgtgaacat 1860 atgtggagtg acgtagcagc ctactttaacctcgacattg aaaacagtga agataataaa 1920 tctactcttt cacttcaatt tgtcgacagcgccgcggata tgccgcttgc gaaaatgcac 1980 ggtgcgtttt caacgaacgt cgtagcaagcaaagaacttc aacagccagg cagtgcacga 2040 agcacgcgac atcttgaaat tgaacttccaaaagaagctt cttatcaaga aggagatcat 2100 ttaggtgtta ttcctcgcaa ctatgaaggaatagtaaacc gtgtaacagc aaggttcggc 2160 ctagatgcat cacagcaaat ccgtctggaagcagaagaag aaaaattagc tcatttgcca 2220 ctcgctaaaa cagtatccgt agaagagcttctgcaatacg tggagcttca agatcctgtt 2280 acgcgcacgc agcttcgcgc aatggctgctaaaacggtct gcccgccgca taaagtagag 2340 cttgaagcct tgcttgaaaa gcaagcctacaaagaacaag tgctggcaaa acgtttaaca 2400 atgcttgaac tgcttgaaaa atacccggcgtgtgaaatga aattcagcga atttatcgcc 2460 cttctgccaa gcatacgccc gcgctattactcgatttctt catcacctcg tgtcgatgaa 2520 aaacaagcaa gcatcacggt cagcgttgtctcaggagaag cgtggagcgg atatggagaa 2580 tataaaggaa ttgcgtcgaa ctatcttgccgagctgcaag aaggagatac gattacgtgc 2640 tttatttcca caccgcagtc agaatttacgctgccaaaag accctgaaac gccgcttatc 2700 atggtcggac cgggaacagg cgtcgcgccgtttagaggct ttgtgcaggc gcgcaaacag 2760 ctaaaagaac aaggacagtc acttggagaagcacatttat acttcggctg ccgttcacct 2820 catgaagact atctgtatca agaagagcttgaaaacgccc aaagcgaagg catcattacg 2880 cttcataccg ctttttctcg catgccaaatcagccgaaaa catacgttca gcacgtaatg 2940 gaacaagacg gcaagaaatt gattgaacttcttgatcaag gagcgcactt ctatatttgc 3000 ggagacggaa gccaaatggc acctgccgttgaagcaacgc ttatgaaaag ctatgctgac 3060 gttcaccaag tgagtgaagc agacgctcgcttatggctgc agcagctaga agaaaaaggc 3120 cgatacgcaa aagacgtgtg ggctgggtaa3150 4 1049 PRT Bacillus megaterium 4 Met Thr Ile Lys Glu Met Pro GlnPro Lys Thr Phe Gly Glu Leu Lys 1 5 10 15 Asn Leu Pro Leu Leu Asn ThrAsp Lys Pro Val Gln Ala Leu Met Lys 20 25 30 Ile Ala Asp Glu Leu Gly GluIle Phe Lys Phe Glu Ala Pro Gly Arg 35 40 45 Val Thr Arg Tyr Leu Ser SerGln Arg Leu Ile Lys Glu Ala Cys Asp 50 55 60 Glu Ser Arg Phe Asp Lys AsnLeu Ser Gln Ala Leu Lys Phe Val Arg 65 70 75 80 Asp Phe Ala Gly Asp GlyLeu Phe Thr Ser Trp Thr His Glu Lys Asn 85 90 95 Trp Lys Lys Ala His AsnIle Leu Leu Pro Ser Phe Ser Gln Gln Ala 100 105 110 Met Lys Gly Tyr HisAla Met Met Val Asp Ile Ala Val Gln Leu Val 115 120 125 Gln Lys Trp GluArg Leu Asn Ala Asp Glu His Ile Glu Val Pro Glu 130 135 140 Asp Met ThrArg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn 145 150 155 160 TyrArg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr 165 170 175Ser Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Leu Gln Arg Ala 180 185190 Asn Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu 195200 205 Asp Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg210 215 220 Lys Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met LeuAsn 225 230 235 240 Gly Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp GluAsn Ile Arg 245 250 255 Tyr Gln Ile Ile Thr Phe Leu Ile Ala Gly His GluThr Thr Ser Gly 260 265 270 Leu Leu Ser Phe Ala Leu Tyr Phe Leu Val LysAsn Pro His Val Leu 275 280 285 Gln Lys Ala Ala Glu Glu Ala Ala Arg ValLeu Val Asp Pro Ala Pro 290 295 300 Ser Tyr Lys Gln Val Lys Gln Leu LysTyr Val Gly Met Val Leu Asn 305 310 315 320 Glu Ala Leu Arg Leu Trp ProThr Ala Pro Ala Phe Ser Leu Tyr Ala 325 330 335 Lys Glu Asp Thr Val LeuGly Gly Glu Tyr Pro Leu Glu Lys Gly Asp 340 345 350 Glu Leu Met Val LeuIle Pro Gln Leu His Arg Asp Lys Thr Ile Trp 355 360 365 Gly Asp Asp ValGlu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser 370 375 380 Ala Ile ProGln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala 385 390 395 400 CysIle Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly 405 410 415Met Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu 420 425430 Asp Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys 435440 445 Ala Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro Ser Thr450 455 460 Glu Gln Ser Ala Lys Lys Ala Arg Lys Lys Ala Glu Asn Ala HisAsn 465 470 475 480 Thr Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly ThrAla Glu Gly 485 490 495 Thr Ala Arg Asp Leu Ala Asp Ile Ala Met Ser LysGly Phe Ala Pro 500 505 510 Gln Val Ala Thr Leu Asp Ser His Ala Gly AsnLeu Pro Arg Glu Gly 515 520 525 Ala Val Leu Ile Val Thr Ala Ser Tyr AsnGly His Pro Pro Asp Asn 530 535 540 Ala Lys Gln Phe Val Asp Trp Leu AspGln Ala Ser Ala Asp Glu Val 545 550 555 560 Lys Gly Val Arg Tyr Ser ValPhe Gly Cys Gly Asp Lys Asn Trp Ala 565 570 575 Thr Thr Tyr Gln Lys ValPro Ala Phe Ile Asp Glu Thr Leu Ala Ala 580 585 590 Lys Gly Ala Glu AsnIle Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp 595 600 605 Asp Phe Glu GlyThr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp 610 615 620 Val Ala AlaTyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys 625 630 635 640 SerThr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu 645 650 655Ala Lys Met His Gly Ala Phe Ser Thr Asn Val Val Ala Ser Lys Glu 660 665670 Leu Gln Gln Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu Ile Glu 675680 685 Leu Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile690 695 700 Pro Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg PheGly 705 710 715 720 Leu Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu GluGlu Lys Leu 725 730 735 Ala His Leu Pro Leu Ala Lys Thr Val Ser Val GluGlu Leu Leu Gln 740 745 750 Tyr Val Glu Leu Gln Asp Pro Val Thr Arg ThrGln Leu Arg Ala Met 755 760 765 Ala Ala Lys Thr Val Cys Pro Pro His LysVal Glu Leu Glu Ala Leu 770 775 780 Leu Glu Lys Gln Ala Tyr Lys Glu GlnVal Leu Ala Lys Arg Leu Thr 785 790 795 800 Met Leu Glu Leu Leu Glu LysTyr Pro Ala Cys Glu Met Lys Phe Ser 805 810 815 Glu Phe Ile Ala Leu LeuPro Ser Ile Arg Pro Arg Tyr Tyr Ser Ile 820 825 830 Ser Ser Ser Pro ArgVal Asp Glu Lys Gln Ala Ser Ile Thr Val Ser 835 840 845 Val Val Ser GlyGlu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile 850 855 860 Ala Ser AsnTyr Leu Ala Glu Leu Gln Glu Gly Asp Thr Ile Thr Cys 865 870 875 880 PheIle Ser Thr Pro Gln Ser Glu Phe Thr Leu Pro Lys Asp Pro Glu 885 890 895Thr Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg 900 905910 Gly Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu 915920 925 Gly Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr930 935 940 Leu Tyr Gln Glu Glu Leu Glu Asn Ala Gln Ser Glu Gly Ile IleThr 945 950 955 960 Leu His Thr Ala Phe Ser Arg Met Pro Asn Gln Pro LysThr Tyr Val 965 970 975 Gln His Val Met Glu Gln Asp Gly Lys Lys Leu IleGlu Leu Leu Asp 980 985 990 Gln Gly Ala His Phe Tyr Ile Cys Gly Asp GlySer Gln Met Ala Pro 995 1000 1005 Ala Val Glu Ala Thr Leu Met Lys SerTyr Ala Asp Val His Gln 1010 1015 1020 Val Ser Glu Ala Asp Ala Arg LeuTrp Leu Gln Gln Leu Glu Glu 1025 1030 1035 Lys Gly Arg Tyr Ala Lys AspVal Trp Ala Gly 1040 1045 5 51 DNA Artificial sequence Primer 5gagattaaga attcataaac acatgggagt gcgtgccata tgaacgcaaa c 51 6 7 PRTArtificial sequence Linker 6 Thr Asp Gly Gly Ser Ser Ser 1 5 7 36 DNAArtificial sequence Desired coding sequence 7 gaactgagta gtgccactgacggaggatcc tcatcg 36 8 5 PRT Artificial sequence Desired coding sequence8 Thr Asp Gly Gly Ser 1 5 9 36 DNA Artificial sequence Primer 9cgatgaggat cctccgtcag tggcactact cagttc 36 10 36 DNA Artificial sequenceDesired coding sequence 10 tcatcgggat cctcatcgat gtctaaagta gtgtat 36 114 PRT Artificial sequence Desired coding sequence 11 Gly Ser Ser Ser 112 52 DNA Artificial sequence Desired coding sequence 12 cccgataggcaatggtaatc atcgggagtc tagagcatcg aagctttcat cg 52 13 52 DNA Artificialsequence Primer 13 cgatgaaagc ttcgatgctc tagactcccg atgattaccattgcctatcg gg 52 14 24 DNA Artificial sequence Primer 14 tcatcgacgcgtcgcgaact gctg 24 15 69 DNA Artificial sequence Desired coding sequence15 cccgataggc aatggtaagt aggtgaatat ctaatcccca tctatgcgcg agtggagtct 60agagttcga 69 16 21 DNA Artificial sequence Primer 16 tcatcgaagcttggctgttt t 21 17 47 DNA Artificial sequence Desired coding sequence 17acaatttcac acaggatcta gaccatatgt catcgaagct ttcatcg 47 18 47 DNAArtificial Sequence Primer 18 cgatgaaagc ttcgatgaca tatggtctagatcctgtgtg aaattgt 47

What is claimed is:
 1. A process for oxidizing a substrate which is1,2-dichlorobenzene, 1, 2, 4-trichlorobenzene, 3, 3′-dichlorobiphenyl,2, 2′, 4, 5, 5′-pentachlorobiphenyl, pentachlorobenzene orhexachlorobenzene, said process comprising the step of oxidizing saidsubstrate with a mutant P450 monooxygenase enzyme in the presence of anelectron transfer reductase and an electron transfer redoxin, whereinthe enzyme is a P450cam enzyme as shown by SEQ ID NO:2, comprising amutation at one or more of the amino acid positions selected from thegroup consisting of 87, 96, 98, 101, 185, 244, 247, 248, 295, 296, 395and 396, and wherein the mutation is a substitution of an amino acidwith an amino acid that has a less polar side chain, and further whereina ring carbon of the substrate is oxidised in the oxidation reaction. 2.The process of claim 1 in which the enzyme comprises a mutation at morethan one of said amino acid positions.
 3. The process of claim 1 inwhich the enzyme comprises a mutation at amino acid 96 of P450_(cam).